N. Kops (Nicole)http://repub.eur.nl/ppl/3291/
List of Publicationsenhttp://repub.eur.nl/eur_signature.pnghttp://repub.eur.nl/
RePub, Erasmus University RepositoryIn vitro model to study the biomaterial-dependent reaction of macrophages in an inflammatory environmenthttp://repub.eur.nl/pub/55976/
Fri, 05 Dec 2014 00:00:01 GMT<div>N. Grotenhuis</div><div>H.F.E. Vd Toom</div><div>N. Kops</div><div>Y. Bayon</div><div>E. Deerenberg</div><div>I.M. Mulder</div><div>G.J.V.M. van Osch</div><div>J.F. Lange</div><div>Y.M. Bastiaansen-Jenniskens</div>
Background Macrophages play an important role in the reaction to biomaterials, which sometimes have to be used in a surgical field at risk of contamination. The macrophage phenotype in reaction to biomaterials in an inflammatory environment was evaluated in both an in vivo and in vitro setting. Methods In the in vivo setting, polypropylene (PP) biomaterial was implanted for 28 days in the contaminated abdominal wall of rats, and upon removal analysed by routine histology as well as immunohistochemistry for CD68 (marker for macrophages), inducible nitric oxide synthase (iNOS - a marker for proinflammatory M1 macrophages) and CD206 (marker for anti-inflammatory M2 macrophages). For the in vitro model, human peripheral blood monocytes were cultured for 3 days on biomaterials made from PP, collagen (COL), polyethylene terephthalate (PET) and PET coated with collagen (PET+COL). These experiments were performed both with and without lipopolysaccharide and interferon γ stimulation. Secretion of both M1- and M2-related proteins was measured, and a relative M1/M2 index was calculated. Results In vivo, iNOS- and CD206-positive cells were found around the fibres of the implanted PP biomaterial. In vitro, macrophages on both PP and COL biomaterial had a relatively low M1/M2 index. Macrophages on the PET biomaterial had a high M1/M2 index, with the highest increase of M1 cytokines in an inflammatory environment. Macrophages on the PET+COL biomaterial also had a high M1/M2 index. Conclusion Macrophages in an inflammatory environment in vitro still react in a biomaterial-dependent manner. This model can help to select biomaterials that are tolerated best in a surgical environment at risk of contamination. Surgical relevance Biomaterials in an environment at risk of contamination are often not tolerated owing to a high risk of postoperative infection, which may ultimately lead to removal of the biomaterial. An in vitro model with primary human macrophages was used to provide insight into the acute reaction of macrophages to a biomaterial in an inflammatory environment simulated with lipopolysaccharide and interferon γ. The reaction of macrophages in such an inflammatory environment was still biomaterial-dependent. This in vitro model can be used to study the reaction of macrophages to different biomaterials in an inflammatory environment in more detail, and thereby help to select biomaterials that are tolerated best in a surgical environment at risk of contamination. Macrophages react mesh dependentlyThe in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilagehttp://repub.eur.nl/pub/77088/
Sun, 06 Apr 2014 00:00:01 GMT<div>M.M. Pleumeekers</div><div>L. Nimeskern</div><div>J.L.M. Koevoet</div><div>N. Kops</div><div>R.M.L. Poublon</div><div>K.S. Stok</div><div>G.J.V.M. van Osch</div>
Abstract
Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. This study evaluated the performance of culture-expanded human chondrocytes from ear (EC), nose (NC) and articular joint (AC), as well as bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells both in vitro and in vivo. All cells (≥ 3 donors per source) were culture-expanded, encapsulated in alginate and cultured for 5 weeks. Subsequently, constructs were implanted subcutaneously for 8 additional weeks. Before and after implantation, glycosaminoglycan (GAG) and collagen content were measured using biochemical assays. Mechanical properties were determined using stress-strain-indentation tests. Hypertrophic differentiation was evaluated with qRT-PCR and subsequent endochondral ossification with histology. ACs had higher chondrogenic potential in vitro than the other cell sources, as assessed by gene expression and GAG content (p < 0.001). However, after implantation, ACs did not further increase their matrix. In contrast, ECs and NCs continued producing matrix in vivo leading to higher GAG content (p < 0.001) and elastic modulus. For NC-constructs, matrix-deposition was associated with the elastic modulus (R² = 0.477, p = 0.039). Although all cells--except ACs--expressed markers for hypertrophic differentiation in vitro, there was no bone formed in vivo. Our work shows that cartilage formation and functionality depends on the cell source used. ACs possess the highest chondrogenic capacity in vitro, while ECs and NCs are most potent in vivo, making them attractive cell sources for cartilage repair.Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defectshttp://repub.eur.nl/pub/77089/
Sun, 09 Feb 2014 00:00:01 GMT<div>J. van der Stok</div><div>M.K.E. Koolen</div><div>H. Jahr</div><div>N. Kops</div><div>J.H. Waarsing</div><div>H.H. Weinans</div><div>O.P. van der Jagt</div>
Abstract:
Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.Chondrogenesis of mesenchymal stem cells in an osteochondral environment is mediated by the subchondral bonehttp://repub.eur.nl/pub/55448/
Wed, 01 Jan 2014 00:00:01 GMT<div>J.R. Vos</div><div>R. Narcisi</div><div>N. Kops</div><div>J.L.M. Koevoet</div><div>P.K. Bos</div><div>J.M. Murphy</div><div>J.A.N. Verhaar</div><div>P.M. van der Kraan</div><div>G.J.V.M. van Osch</div>
In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFβ) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFβ; however, the observed results could not be attributed to TGFβ, as either capturing TGFβ with an antibody or blocking the canonical TGFβ signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFβ family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.Mesenchymal stem cells reduce pain but not degenerative changes in a mono-iodoacetate rat model of osteoarthritishttp://repub.eur.nl/pub/69939/
Wed, 01 Jan 2014 00:00:01 GMT<div>G.M. van Buul</div><div>M. Siebelt</div><div>M.J.C. Leijs</div><div>P.K. Bos</div><div>J.H. Waarsing</div><div>N. Kops</div><div>H.H. Weinans</div><div>J.A.N. Verhaar</div><div>M.R. Bernsen</div><div>G.J.V.M. van Osch</div>
We studied the effects of intra-articularly injected bone marrow derived mesenchymal stem cells (MSCs), as well as freshly isolated bone marrow mononuclear cells (BMMNCs), on pain, cartilage damage, bone changes and inflammation in an in-vivo rat osteoarthritis (OA) model. OA was induced unilaterally by injection of mono-iodoacetate (MIA) and allowed to develop for 3 weeks. Then, animals were treated by intra-articular injection with MSCs, BMMNCs, or saline as a control. Four weeks later, pain was assessed with an incapitance tester, subchondral bone alterations were measured with μCT and cartilage quality and joint inflammation were assessed by histological analysis. Animals treated with MSCs distributed significantly more weight to the affected limb after treatment, which was not observed in the other groups. No statistically significant differences between treatment groups regarding cartilage damage, subchondral bone alterations and synovial inflammation were observed. Additional cell tracking experiments indicated adequate intra-articular cell injection and cell survival up to 2 weeks. In our OA model, injected MSCs were able to reduce MIA induced pain, as measured by an increased weight distribution to the affected limb. No statistically significant effects of the cellular therapies on structural damage and synovial inflammation were found.Properties of commonly used calcium phosphate cements in trauma and orthopaedic surgeryhttp://repub.eur.nl/pub/53827/
Tue, 01 Oct 2013 00:00:01 GMT<div>J. van der Stok</div><div>H.H. Weinans</div><div>N. Kops</div><div>M. Siebelt</div><div>P. Patka</div><div>E.M.M. van Lieshout</div>
Hsp90 inhibition protects against biomechanically induced osteoarthritis in ratshttp://repub.eur.nl/pub/59249/
Thu, 01 Aug 2013 00:00:01 GMT<div>M. Siebelt</div><div>H. Jahr</div><div>H.C. Groen</div><div>M. Sandker</div><div>J.H. Waarsing</div><div>N. Kops</div><div>C. Müller</div><div>W. van Eden</div><div>M. de Jong</div><div>H.H. Weinans</div>
Objective Although articular cartilage has evolved to facilitate joint mobilization, severe loading can induce chondrocyte apoptosis, which is related to the progression of osteoarthritis (OA). To avoid apoptosis, chondrocytes synthesize heat-shock proteins (HSPs). This study was undertaken to examine the roles of Hsp70 and Hsp90 in biomechanically induced OA, and the possibility of using Hsp90 inhibition as an intervention strategy for OA management. Methods OA was biomechanically induced in rats by means of strenuous running. Disease progression was compared between running rats treated with Hsp90 inhibitor and untreated running controls. Hsp70 and Hsp90 protein levels in articular cartilage were determined by Western blotting. OA progression was monitored using contrast-enhanced micro-computed tomography to measure cartilage degradation and subchondral bone changes and single-photon-emission computed tomography to examine synovial macrophage activation and histologic features. Results Chronic cartilage loading led to early OA development, characterized by degeneration of cartilage extracellular matrix. In vivo Hsp90 inhibition resulted in increased Hsp70 synthesis, which suggests that Hsp90 activity limits Hsp70 production. Hsp90 inhibitor treatment increased cartilage sulfated glycosaminoglycan levels to concentrations even beyond baseline and protected against cartilage degradation, stimulated subchondral bone thickness, and suppressed macrophage activation. Conclusion Our findings indicate that Hsp90 plays a pivotal role in biomechanically induced chondrocyte stress responses. Intervention strategies that inhibit Hsp90 can potentially protect or improve cartilage health and might prevent OA development. CopyrightUnfocused extracorporeal shock waves induce anabolic effects in osteoporotic ratshttp://repub.eur.nl/pub/39606/
Wed, 01 May 2013 00:00:01 GMT<div>O.P. van der Jagt</div><div>J.H. Waarsing</div><div>N. Kops</div><div>W. Schaden</div><div>H. Jahr</div><div>J.A.N. Verhaar</div><div>H.H. Weinans</div>
Unfocused extracorporeal shock waves (UESW) have been shown to have an anabolic effect on bone mass. Therefore we investigated the effects of UESW on bone in osteoporotic rats with and without anti-resorptive treatment. Twenty-week-old rats were ovariectomized (n = 27). One group was treated with saline and another group with Alendronate (ALN) 2.4 μg/kg, 3×/week. UESW were applied 2 weeks after ovariectomy. Thousand UESW were applied to one hind leg, the contra-lateral hind leg was not treated and served as control. With the use of in vivo micro-CT scanning it was shown that in saline treated rats trabecular bone volume fraction (BV/TV) was higher at 2 weeks follow-up in UESW treated legs compared to control legs. However, at 4 and 10 weeks no difference was found. In ALN treated animals UESW led to a pronounced anabolic response resulting in an increase in BV/TV at all time-points. Furthermore, UESW resulted in increased cortical volume (CtV), higher trabecular connectivity and, more plate-like and thicker trabeculae. Biomechanical testing showed that UESW lead to a higher maximum force before failure and higher stiffness in all treatment groups. With histology abundant areas of intramembranous bone formation along the periosteal cortex and within the bone marrow were observed. In conclusion this study shows promising results for the use of UESW in the treatment of osteoporosis, especially when this treatment is combined with an anti-resorptive treatment. Copyright Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culturehttp://repub.eur.nl/pub/39233/
Mon, 01 Oct 2012 00:00:01 GMT<div>G.M. van Buul</div><div>E. Villafuertes</div><div>P.K. Bos</div><div>J.H. Waarsing</div><div>N. Kops</div><div>R. Narcisi</div><div>H.H. Weinans</div><div>J.A.N. Verhaar</div><div>M.R. Bernsen</div><div>G.J.V.M. van Osch</div>
Objective: Mesenchymal stem cells (MSCs) are promising candidates for osteoarthritis (OA) therapies, although their mechanism of action remains unclear. MSCs have recently been discovered to secrete anti-inflammatory cytokines and growth factors. We studied the paracrine effects of MSCs on OA cartilage and synovial explants in vitro. Design: MSC-conditioned medium was prepared by stimulating primary human MSCs with tumour necrosis factor alpha (TNFα) and (50. ng/ml each). Human synovium and cartilage explants were cultured in MSC-conditioned medium or in control medium, containing the same amount of added TNFα and IFNγ but not incubated with MSCs. Explants were analyzed for gene expression and the production of nitric oxide (NO). The presence of the inhibitor of nuclear factor kappa B alpha (IκBa) was assessed by Western blot analysis. Results: Synovial explants exposed to MSC-conditioned medium showed decreased gene expression of interleukin-1 beta (IL-1β), matrix metalloproteinase (MMP). 1 and MMP13, while suppressor of cytokine signaling (SOCS). 1 was upregulated. In cartilage, expression of IL-1 receptor antagonist (IL-1RA) was upregulated, whereas a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). 5 and collagen type II alpha 1 (COL2A1) were downregulated. MSC-conditioned medium reduced NO production in cartilage explants and the presence of IκBa was increased in synoviocytes and chondrocytes treated with MSC-conditioned medium. Conclusions: In an inflammatory environment, MSCs secrete factors which cause multiple anti-inflammatory effects and influence matrix turnover in synovium and cartilage explants. Thereby, the presented data encourage further study of MSCs as a treatment for joint diseases. Inhibiting calcineurin activity under physiologic tonicity elevates anabolic but suppresses catabolic chondrocyte markershttp://repub.eur.nl/pub/39373/
Fri, 01 Jun 2012 00:00:01 GMT<div>A.E. van der Windt</div><div>E. Haak</div><div>N. Kops</div><div>J.A.N. Verhaar</div><div>H.H. Weinans</div><div>H. Jahr</div>
Objective The physiologic interstitial tonicity of healthy articular cartilage (350-480 mOsm) is lowered to 280-350 mOsm in osteoarthritis (OA). This results in loss of tissue prestress, altered compressive behavior, and, thus, inferior tissue properties. This study was undertaken to determine whether physiologic tonicity in combination with the inhibition of calcineurin (Cn) activity by FK-506 has synergistic effects on human articular chondrocytes and explants in vitro. Methods OA chondrocytes and explants and non-OA chondrocytes were cultured in cytokine-free medium of 280 mOsm or 380 mOsm with or without Cn inhibition by FK-506. Chondrogenic, hypertrophic, and catabolic marker expression was evaluated at the messenger RNA (mRNA), protein, and activity levels. Results Compared to OA chondrocytes cultured at 280 mOsm, those cultured at 380 mOsm had increased expression of mRNA for chondrogenic markers (e.g., ∼13 fold for COL2; P < 0.001), and decreased COL1 expression (∼0.5 fold, P < 0.01). Inhibiting Cn activity under physiologic tonicity further enhanced the expression of anabolic markers at the mRNA level (∼50 fold for COL2; P < 0.001, ∼2 fold for AGC1; P < 0.001, and ∼3.5 fold for SOX9; P < 0.001) and at the protein level (∼6 fold for type II collagen; P < 0.001). Cn inhibition suppressed relevant collagenases as well as hypertropic and mineralization markers at the mRNA and activity levels. Expression of aggrecanase 1 and aggrecanase 2 was not influenced by tonicity or FK-506 alone, but the combination suppressed both, by ∼50% (P < 0.05) and ∼40% (P < 0.001), respectively. Generally, similar anabolic and antihypertrophic effects were observed in ex vivo cartilage explant cultures and non-OA chondrocytes. Conclusion Our findings indicate that Cn at physiologic tonicity exerts a superior effect compared to physiologic tonicity or FK-506 alone, increasing anabolic markers while suppressing hypertrophic and catabolic markers. Our data may aid in the development of improved cell-based chondral repair and OA treatment strategies. Copyright An osteochondral culture model to study mechanisms involved in articular cartilage repairhttp://repub.eur.nl/pub/34920/
Sun, 01 Jan 2012 00:00:01 GMT<div>M.L. de Vries-Van Melle</div><div>E.W. Mandl</div><div>N. Kops</div><div>J.L.M. Koevoet</div><div>J.A.N. Verhaar</div><div>G.J.V.M. van Osch</div>
Although several treatments for cartilage repair have been developed and used in clinical practice the last 20 years, little is known about the mechanisms that are involved in the formation of repair tissue after these treatments. Often, these treatments result in the formation of fibrocartilaginous tissue rather than normal articular cartilage. Because the repair tissue is inferior to articular cartilage in terms of mechanical properties and zonal organization of the extracellular matrix, complaints of the patient may return. The biological and functional outcome of these treatments should thus be improved. For this purpose, an in vitro model allowing investigation of the involved repair mechanisms can be of great value. We present the development of such a model. We used bovine osteochondral biopsies and created a system in which cartilage defects of different depths can be studied. First, our biopsy model was characterized extensively: we studied the viability by means of lactate dehydrogenase (LDH) excretion over time and we investigated expression of cartilage-related genes in osteochondral biopsies and compared it with conventional cartilage-only explants. After 28 days of culture, LDH was detected at low levels and mRNA could be retrieved. The expression of cartilage-related genes decreased over time. This was more evident in cartilage-only explants, indicating that the biopsy model provided a more stable environment. We also characterized the subchondral bone: osteoclasts and osteoblasts were active after 28 days of culture, which was indicated by tartrate acid phosphatase staining and alkaline phosphatase measurements, respectively, and matrix deposition during culture was visualized using calcein labeling. Second, the applicability of the model was further studied by testing two distinct settings: (1) implantation of chondrocytes in defects of different depths; (2) two different seeding strategies of chondrocytes. Differences were observed in terms of volume and integration of newly formed tissue in both settings, suggesting that our model can be used to model distinct conditions or even to mimic clinical treatments. After extensive characterization and testing of our model, we present a representative and reproducible in vitro model that can be used to evaluate new cartilage repair treatments and study mechanisms in a controlled and standardized environment. Platelet-rich plasma releasate inhibits inflammatory processes in osteoarthritic chondrocyteshttp://repub.eur.nl/pub/33820/
Tue, 01 Nov 2011 00:00:01 GMT<div>G.M. van Buul</div><div>J.L.M. Koevoet</div><div>N. Kops</div><div>P.K. Bos</div><div>J.A.N. Verhaar</div><div>H.H. Weinans</div><div>M.R. Bernsen</div><div>G.J.V.M. van Osch</div>
Background: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes.Hypothesis: Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes.Study Design: Controlled laboratory study.Methods: Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFB) activation were studied.Results: Platelet-rich plasma releasate diminished IL-1 beta-induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta-induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta-induced NFB activation to control levels containing no IL-1 beta.Conclusion: Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta-mediated effects on human osteoarthritic chondrocytes, including inhibition of NFB activation.Clinical Relevance: Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA. Quantifying osteoarthritic cartilage changes accurately using in vivo microCT arthrography in three etiologically distinct rat modelshttp://repub.eur.nl/pub/33899/
Tue, 01 Nov 2011 00:00:01 GMT<div>M. Siebelt</div><div>J.H. Waarsing</div><div>N. Kops</div><div>T.M. Piscaer</div><div>J.A.N. Verhaar</div><div>E.H.G. Oei</div><div>H.H. Weinans</div>
In vivo microCT arthrography (μCTa) can be used to measure both quantity (volumetric) and quality (glycosaminoglycan content) of cartilage. This study investigated the accuracy of four segmentation techniques to isolate cartilage from μCTa datasets and then used the most accurate one to investigate if the μCTa method could show osteoarthritic changes in rat models during longitudinal follow-up. Volumetric measurements and glycosaminoglycan contents of patellar cartilage from in vivo μCTa-scans were compared with an ex vivo gold standard μCT-scan. Cartilage was segmented with three global thresholds and one local threshold algorithm. Comparisons were made for healthy and osteoarthritic cartilage. Next, three rat models were investigated for 24 weeks using μCTa. Osteoarthritis was induced by injection with a chemical (mono-iodoacetate), a surgical intervention (grooves applied in articular cartilage), and via exercise (strenuous running). After euthanasia, all knee joints were isolated for histology. Local thresholds accurately segmented cartilage from in vivo μCTa scans and best measured cartilage quantity and glycosaminoglycan content. Each of the three osteoarthritic rat models showed a specific pattern of osteoarthritis progression. All μCTa results were comparable to histology. In vivo μCTa is a sensitive technique for imaging cartilage degradation. Local thresholds enhanced the sensitivity of this method and will probably more accurately detect disease-modulating effects from interventional strategies. The data from rat models may serve as a reference for the time sequence of cartilage degeneration during in vivo testing of new strategies in osteoarthritis treatment. Copyright In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cellshttp://repub.eur.nl/pub/23692/
Wed, 02 Feb 2011 00:00:01 GMT<div>E. Farrell</div><div>S.K. Both</div><div>K.I. Odörfer</div><div>J.L.M. Koevoet</div><div>N. Kops</div><div>F.J. O'Brien</div><div>R.J. Baatenburg de Jong</div><div>J.A.N. Verhaar</div><div>V. Cuijpers</div><div>J.A. Jansen</div><div>R.G. Erben</div><div>G.J.V.M. van Osch</div>
Abstract
Background: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients’ own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway.
Methods: MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture.
Results: After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of b-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.
Conclusions: The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of b-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.Unfocused Extracorporeal Shock Waves Induce Anabolic Effects in Rat Bonehttp://repub.eur.nl/pub/23728/
Wed, 05 Jan 2011 00:00:01 GMT<div>O.P. van der Jagt</div><div>T.M. Piscaer</div><div>W. Schaden</div><div>J. Li</div><div>N. Kops</div><div>H. Jahr</div><div>J.C. van der Linden</div><div>J.H. Waarsing</div><div>J.A.N. Verhaar</div><div>M. de Jong</div><div>H.H. Weinans</div>
Abstract. BACKGROUND: Extracorporeal shock waves are known to stimulate the differentiation of mesenchymal stem cells toward osteoprogenitors and induce the expression of osteogenic-related growth hormones. The aim of this study was to investigate if and how extracorporeal shock waves affected new bone formation, bone microarchitecture, and the mechanical properties of bone in a healthy rat model, in order to evaluate whether extracorporeal shock wave therapy might be a potential treatment for osteoporosis.
METHODS: Thirteen rats received 1000 electrohydraulically generated unfocused extracorporeal shock waves to the right tibia. The contralateral, left tibia was not treated and served as a control. At two, seven, twenty-one, and forty-nine days after administration of the shock waves, in vivo single-photon-emission computed tomography (SPECT) scanning was performed to measure new bone formation on the basis of uptake of technetium-labeled methylene diphosphonate ((99m)Tc-MDP) (n = 6). Prior to and forty-nine days after the extracorporeal shock wave therapy, micro-computed tomography (micro-CT) scans were made to examine the architectural bone changes. In addition, mechanical testing, microcrack, and histological analyses were performed.
RESULTS: Extracorporeal shock waves induced a strong increase in (99m)Tc-MDP uptake in the treated tibia compared with the uptake in the untreated, control tibia. Micro-CT analysis showed that extracorporeal shock waves stimulated increases in both trabecular and cortical volume, which resulted in higher bone stiffness compared with that of the control tibiae. Histological analysis showed intramedullary soft-tissue damage and de novo bone with active osteoblasts and osteoid in the bone marrow of the legs treated with extracorporeal shock waves. Microcrack analysis showed no differences between the treated and control legs.
CONCLUSIONS: This study shows that a single treatment with extracorporeal shock waves induces anabolic effects in both cancellous and cortical bone, leading to improved biomechanical properties. Furthermore, treatment with extracorporeal shock waves results in transient damage to the bone marrow, which might be related to the anabolic effects. After further examination and optimization, unfocused extracorporeal shock waves might enable local treatment of skeletal sites susceptible to fracture.Laryngotracheal reconstruction with porous titanium in rabbits: Are vascular carriers and mucosal grafts really necessary?http://repub.eur.nl/pub/20658/
Thu, 01 Jul 2010 00:00:01 GMT<div>L.M. Janssen</div><div>G.J.V.M. van Osch</div><div>N. Kops</div><div>K. de Groot</div><div>L. Feenstra</div><div>J.A.U. Hardillo</div>
Laryngotracheal reconstruction requires a supportive structure with a mucosal lining, which needs a vascular supply in order to regenerate properly. We investigated the necessity of a vascular carrier and mucosal graft when using porous titanium for laryngotracheal reconstruction. Surgical defects of the laryngotracheal complex in 22 rabbits were reconstructed with: (a) porous titanium implanted on a vascularized fascia combined with a buccal mucosal graft (first stage) before transposing to the neck area (second stage); (b) porous titanium implanted on a vascularized fascia (first stage) combined with a mucosal graft (second stage); (c) porous titanium on a pedicled fascia flap; and (d) porous titanium alone. The grafts were tolerated well. Re-epithelialization occurred in all groups. Normal mucosa with a submucosal layer containing vital cells was noted using the titanium implants. Blood vessels were grown in the pores of the titanium scaffold to supply the overlying mucosa. The scaffold was well integrated in the adjacent tracheal cartilage and surrounding tissues, except in the two cases that showed titanium displacement. Inflammation and granulation formation were seen in most rabbits in groups III and IV, initiated probably by the use of buccal mucosal grafts. Reconstruction of a rabbit's trachea using composites of porous titanium, mucosal grafts and a fascia flap is feasible. Titanium seems to meet the requirements needed for closing a small defect of the tracheal wall and allows for re-epithelialization. For larger defects, a vascular carrier with a mucosal graft is probably indispensable to ensure the process of re-epithelialization.Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitrohttp://repub.eur.nl/pub/20665/
Sat, 01 May 2010 00:00:01 GMT<div>A.E. van der Windt</div><div>E. Haak</div><div>R.H.J. Das</div><div>N. Kops</div><div>T.J.M. Welting</div><div>M.M.J. Caron</div><div>N.P. van Til</div><div>J.A.N. Verhaar</div><div>H.H. Weinans</div><div>H. Jahr</div>
Abstract
Introduction: Chondrocytes experience a hypertonic environment compared to plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes
removes their hypertonic matrix, exposing them to non-physiological conditions. During in-vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in-vitro expansion on chondrocyte phenotype.
Methods: Human articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological, tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated.
The role of Tonicity-responsive Enhancer Binding Protein (TonEBP/NFAT5) in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.
Results: Moderately elevated, physiological, tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and ‘normal’ (non-osteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.
Conclusions: Physiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in-vitro expansion of human articular
chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: Relating tissue engineering using adult mesenchymal stem cells to embryonic developmenthttp://repub.eur.nl/pub/20003/
Mon, 01 Feb 2010 00:00:01 GMT<div>C.A. Hellingman</div><div>J.L.M. Koevoet</div><div>N. Kops</div><div>E. Farrell</div><div>H. Jahr</div><div>W. Liu</div><div>R.J.B. de Jong</div><div>D.A. Frenz</div><div>G.J.V.M. van Osch</div>
Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.Ferumoxides–protamine sulfate is more effective than ferucarbotran for cell labeling: implications for clinically applicable cell tracking using MRIhttp://repub.eur.nl/pub/17601/
Fri, 01 Jan 2010 00:00:01 GMT<div>G.M. van Buul</div><div>E. Farrell</div><div>N. Kops</div><div>S.T. van Tiel</div><div>P.K. Bos</div><div>H.H. Weinans</div><div>G.P. Krestin</div><div>G.J.V.M. van Osch</div><div>M.R. Bernsen</div>
The use of superparamagnetic iron oxide (SPIO) for labeling cells holds great promise for clinically applicable cell tracking using magnetic resonance imaging. For clinical application, an effectively and specifically labeled cell preparation is highly desired (i.e. a large amount of intracellular iron and a negligible amount of extracellular iron). In this study we performed a direct comparison of two SPIO labeling strategies that have both been reported as efficient and clinically translatable approaches. These approaches are cell labeling using ferumoxides-protamine complexes or ferucarabotran particles. Cell labeling was performed on primary human bone marrow stromal cells (hBMSCs) and chondrocytes. For both cell types ferumoxides-protamine resulted in a higher percentage of labeled cells, a higher total iron load, a larger amount of intracellular iron and a lower amount of extracellular iron aggregates, compared with ferucarbotran. Consequently, hBMSC and chondrocyte labeling with ferumoxides-protamine is more effective and results in more specific cell labeling than ferucarbotran.Cell labelling with superparamagnetic iron oxide has no effect on chondrocyte behaviourhttp://repub.eur.nl/pub/15302/
Wed, 01 Jul 2009 00:00:01 GMT<div>E. Farrell</div><div>P.A. Wielopolski</div><div>P. Pavljasevic</div><div>N. Kops</div><div>H.H. Weinans</div><div>M.R. Bernsen</div><div>G.J.V.M. van Osch</div>
BACKGROUND: Tissue engineering and regenerative medicine are two rapidly advancing fields of research offering potential for effective treatment of cartilage lesions. Today, chondrocytes are the cell type of choice for use in cartilage repair approaches such as autologous chondrocyte implantation. To verify the safety and efficacy of such approaches it is necessary to determine the fate of these transplanted cells. One way of doing this is prelabelling cells before implantation and tracking them using imaging techniques. The use of superparamagnetic iron oxide (SPIO) for tracking of cells with magnetic resonance imaging (MRI) is ideal for this purpose. It is non-radioactive, does not require viral transfection and is already approved for clinical use as a contrast agent. OBJECTIVE: The purpose of this study was to assess the effect of SPIO labelling on adult human chondrocyte behaviour. METHODS: Cells were culture expanded and dedifferentiated for two passages and then labelled with SPIO. Effect on cell proliferation was tested. Furthermore, cells were cultured for 21 days in alginate beads in redifferentiation medium. Following this period, cells were analysed for expression of cartilage-related genes, proteoglycan production and collagen protein expression. RESULTS: SPIO labelling did not significantly affect any of these parameters relative to unlabelled controls. We also demonstrated SPIO retention within the cells for the full duration of the experiment. CONCLUSIONS: This paper demonstrates for the first time the effects of SPIO labelling on chondrocyte behaviour, illustrating its potential for in vivo tracking of implanted chondrocytes.